The KDM6A-KMT2D-p300 axis regulates susceptibility to diverse coronaviruses by mediating viral receptor expression.

Identification of host determinants of coronavirus infection informs mechanisms of pathogenesis and may provide novel therapeutic targets. Here, we demonstrate that the histone demethylase KDM6A promotes infection of diverse coronaviruses, including SARS-CoV, SARS-CoV-2, MERS-CoV and mouse hepatitis virus (MHV) in a demethylase activity-independent manner. Mechanistic studies reveal that KDM6A promotes viral entry by regulating expression of multiple coronavirus receptors, including ACE2, DPP4 and Ceacam1. Importantly, the TPR domain of KDM6A is required for recruitment of the histone methyltransferase KMT2D and histone deacetylase p300. Together this KDM6A-KMT2D-p300 complex localizes to the proximal and distal enhancers of ACE2 and regulates receptor expression. Notably, small molecule inhibition of p300 catalytic activity abrogates ACE2 and DPP4 expression and confers resistance to all major SARS-CoV-2 variants and MERS-CoV in primary human airway and intestinal epithelial cells. These data highlight the role for KDM6A-KMT2D-p300 complex activities in conferring diverse coronaviruses susceptibility and reveal a potential pan-coronavirus therapeutic target to combat current and emerging coronaviruses. One Sentence Summary: The KDM6A/KMT2D/EP300 axis promotes expression of multiple viral receptors and represents a potential drug target for diverse coronaviruses.

Transverse myelitis following measles vaccination in a rhesus macaque (Macaca mulatta).

A 16-year-old rhesus macaque presented with progressive, ascending quadriparesis following measles vaccination. He was diagnosed with transverse myelitis following MRI, gross necropsy, and histopathology. This is the first report of transverse myelitis in a rhesus macaque following measles vaccination.

Preimplantation factor reduces graft-versus-host disease by regulating immune response and lowering oxidative stress (murine model).

Bone marrow transplantation (BMT) to treat severe hematologic malignancies often leads to potentially fatal acute graft-versus-host disease (GVHD), despite attempts at better donor-recipient matching and/or use of immunosuppressive agents. We report that embryo-derived PreImplantation Factor (PIF) plays a determining role in developing maternal/host tolerance toward the semiallogeneic or total allogeneic embryo and in regulating systemic immune response. Synthetic PIF treatment has proven effective in preventing immune attacks in nonpregnant models of autoimmunity. In this study, we tested the capability of PIF to prevent the development of acute GVHD in semiallogeneic or totally allogeneic murine BMT models. We examined the regulatory effect of PIF both in vivo and in vitro to control deleterious GVHD while maintaining its ability to preserve the beneficial graft-versus-leukemia (GVL) effect. Bone marrow and spleen cells from C57BL/6 donors were transplanted in semiallogeneic (C57BL/6xBALB/c) F1 or allogeneic (BALB/c) mice, which were then treated with PIF 1 mg/kg/day for 2 weeks. Short-term PIF administration reduced acute GVHD in both models and increased survival for up to 4 months after semiallogeneic or totally allogeneic BMT. This effect was coupled with decreased skin inflammation (semiallogeneic model) and decreased liver inflammation (both models), as well as reduced colon ulceration (allogeneic model). GVHD-associated cytokine and chemokine gene expression were decreased in the liver. PIF further lowered circulating IL-17 levels, but not IFN-γ levels. Both in vivo and in vitro, PIF treatment was demonstrated to lead to decreased inducible nitric oxide synthase expression and decreased lipopolysaccharide-activated macrophages to lower nitric oxide secretion. Significantly, PIF did not diminish the beneficial GVL effect in the B cell leukemia model. PIF acts primarily by inducing the regulatory phenotype on monocytes/antigen-presenting cells, which controls T cell proliferation. Overall, our data demonstrate that PIF protects against semiallogeneic and allogeneic GVHD long term by reducing both target organ and systemic inflammation and by decreasing oxidative stress, while preserving the beneficial GVL effect.

Modeling pandemic to endemic patterns of SARS-CoV-2 transmission using parameters estimated from animal model data.

The contours of endemic coronaviral disease in humans and other animals are shaped by the tendency of coronaviruses to generate new variants superimposed upon nonsterilizing immunity. Consequently, patterns of coronaviral reinfection in animals can inform the emerging endemic state of the SARS-CoV-2 pandemic. We generated controlled reinfection data after high and low risk natural exposure or heterologous vaccination to sialodacryoadenitis virus (SDAV) in rats. Using deterministic compartmental models, we utilized in vivo estimates from these experiments to model the combined effects of variable transmission rates, variable duration of immunity, successive waves of variants, and vaccination on patterns of viral transmission. Using rat experiment-derived estimates, an endemic state achieved by natural infection alone occurred after a median of 724 days with approximately 41.3% of the population susceptible to reinfection. After accounting for translationally altered parameters between rat-derived data and human SARS-CoV-2 transmission, and after introducing vaccination, we arrived at a median time to endemic stability of 1437 (IQR = 749.25) days with a median 15.4% of the population remaining susceptible. We extended the models to introduce successive variants with increasing transmissibility and included the effect of varying duration of immunity. As seen with endemic coronaviral infections in other animals, transmission states are altered by introduction of new variants, even with vaccination. However, vaccination combined with natural immunity maintains a lower prevalence of infection than natural infection alone and provides greater resilience against the effects of transmissible variants.

Natural rodent model of viral transmission reveals biological features of virus population dynamics.

Emerging viruses threaten global health, but few experimental models can characterize the virus and host factors necessary for within- and cross-species transmission. Here, we leverage a model whereby pet store mice or rats-which harbor natural rodent pathogens-are cohoused with laboratory mice. This "dirty" mouse model offers a platform for studying acute transmission of viruses between and within hosts via natural mechanisms. We identified numerous viruses and other microbial species that transmit to cohoused mice, including prospective new members of the Coronaviridae, Astroviridae, Picornaviridae, and Narnaviridae families, and uncovered pathogen interactions that promote or prevent virus transmission. We also evaluated transmission dynamics of murine astroviruses during transmission and spread within a new host. Finally, by cohousing our laboratory mice with the bedding of pet store rats, we identified cross-species transmission of a rat astrovirus. Overall, this model system allows for the analysis of transmission of natural rodent viruses and is a platform to further characterize barriers to zoonosis.

Overview of Coronaviruses in Veterinary Medicine.

Coronaviruses infect humans and a wide range of animals, causing predominantly respiratory and intestinal infections. This review provides background on the taxonomy of coronaviruses, the functions of viral proteins, and the life cycle of coronaviruses. In addition, the review focuses on coronaviral diseases in several agriculturally important, companion, and laboratory animal species (cats, cattle, chickens, dogs, mice, rats and swine) and briefly reviews human coronaviruses and their origins.

Modeling SARS-CoV-2 propagation using rat coronavirus-associated shedding and transmission.

At present, global immunity to SARS-CoV-2 resides within a heterogeneous combination of susceptible, naturally infected and vaccinated individuals. The extent to which viral shedding and transmission occurs on re-exposure to SARS-CoV-2 is an important determinant of the rate at which COVID-19 achieves endemic stability. We used Sialodacryoadenitis Virus (SDAV) in rats to model the extent to which immune protection afforded by prior natural infection via high risk (inoculation; direct contact) or low risk (fomite) exposure, or by vaccination, influenced viral shedding and transmission on re-exposure. On initial infection, we confirmed that amount, duration and consistency of viral shedding, and seroconversion rates were correlated with exposure risk. Animals were reinfected after 3.7-5.5 months using the same exposure paradigm. 59% of seropositive animals shed virus, although at lower amounts. Previously exposed seropositive reinfected animals were able to transmit virus to 25% of naive recipient rats after 24-hour exposure by direct contact. Rats vaccinated intranasally with a related virus (Parker's Rat Coronavirus) were able to transmit SDAV to only 4.7% of naive animals after a 7-day direct contact exposure, despite comparable viral shedding. Cycle threshold values associated with transmission in both groups ranged from 29-36 cycles. Observed shedding was not a prerequisite for transmission. Results indicate that low-level shedding in both naturally infected and vaccinated seropositive animals can propagate infection in susceptible individuals. Extrapolated to COVID-19, our results suggest that continued propagation of SARS-CoV-2 by seropositive previously infected or vaccinated individuals is possible.

PCR and RT-PCR in the Diagnosis of Laboratory Animal Infections and in Health Monitoring.

Molecular diagnostics (PCR and RT-PCR) have become commonplace in laboratory animal research and diagnostics, augmenting or replacing serological and microbiologic methods. This overview will discuss the uses of molecular diagnostics in the diagnosis of pathogenic infections of laboratory animals and in monitoring the microbial status of laboratory animals and their environment. The article will focus primarily on laboratory rodents, although PCR can be used on samples from any laboratory animal species.

Cutaneous and Pulmonary Mucormycosis in Rag1- and Il2rg-deficient Rats.

Immunodeficient rats are valuable in transplantation studies, but are vulnerable to infection from opportunistic organisms such as fungi. Immunodeficient Rag1- and Il2rg-deficient (RRG) rats housed at our institution presented with dark, proliferative, keratinized dermal growths. Histologic and PCR results indicated that the predominant organism associated with these lesions was fungus from the family Mucoraceae, mostly of the genus Rhizopus. The Mucoraceae family of fungi are environmental saprophytes and are often found in rodent bedding. These fungi can cause invasive opportunistic infections in immunosuppressed humans and animals. We discuss husbandry practices for immunosuppressed rodents with a focus on controlling fungal contaminants.

Human PI3Kγ deficiency and its microbiota-dependent mouse model reveal immunodeficiency and tissue immunopathology.

Phosphatidylinositol 3-kinase-gamma (PI3Kγ) is highly expressed in leukocytes and is an attractive drug target for immune modulation. Different experimental systems have led to conflicting conclusions regarding inflammatory and anti-inflammatory functions of PI3Kγ. Here, we report a human patient with bi-allelic, loss-of-function mutations in PIK3CG resulting in absence of the p110γ catalytic subunit of PI3Kγ. She has a history of childhood-onset antibody defects, cytopenias, and T lymphocytic pneumonitis and colitis, with reduced peripheral blood memory B, memory CD8+ T, and regulatory T cells and increased CXCR3+ tissue-homing CD4 T cells. PI3Kγ-deficient macrophages and monocytes produce elevated inflammatory IL-12 and IL-23 in a GSK3α/ß-dependent manner upon TLR stimulation. Pik3cg-deficient mice recapitulate major features of human disease after exposure to natural microbiota through co-housing with pet-store mice. Together, our results emphasize the physiological importance of PI3Kγ in restraining inflammation and promoting appropriate adaptive immune responses in both humans and mice.

Reliability of soiled bedding transfer for detection of mouse parvovirus and mouse hepatitis virus.

Serologic monitoring of sentinel mice exposed to soiled bedding is a common method of detecting viral infections in mice. Because bedding transfer protocols vary, the sensitivity of this method has not been documented sufficiently. We examined the reliability of bedding transfer during various stages of infection with mouse parvovirus (MPV) and mouse hepatitis virus (MHV). Most mice exposed to bedding contaminated with MPV 0, 3, or 7 d previously seroconverted, whereas only mice exposed to bedding contaminated with MHV 4 h previously seroconverted, thus confirming the differing stabilities of these viruses. Index mice were inoculated with 30 times the infectious dose 50 (ID50) of MPV or 300 ID50 of MHV. At 3 d, 1 wk, and 2 wk postinoculation (PI), we transferred 25, 50, or 100 ml of bedding to cages of sentinel mice. Viral infection and shedding by index mice was confirmed by serology and fecal polymerase chain reaction assay. Transfer of soiled bedding between mice in static cages induced seroconversion of sentinel mice most reliably during peak viral shedding (1 wk PI for MPV and 3 d PI for MHV). Soiled bedding transfer between mice in individually ventilated cages induced a higher prevalence of sentinel seroconversion to MPV and MHV than that after transfer between mice in static cages. Our findings indicate that although soiled bedding transfer is an effective method for detecting MHV and MPV under optimal conditions, the method is less than 100% reliable under many conditions in contemporary mouse facilities.

Lack of Effect of Murine Astrovirus Infection on Dextran Sulfate-induced Colitis in NLRP3-deficient Mice.

Murine astrovirus (MuAstV) is a recently identified, widespread infection among laboratory mice. MuAstV is found predominantly in the gastrointestinal tract of mice. Human and turkey astroviruses have been shown to disrupt tight junctions in the intestinal epithelium. The potential of MuAstV to alter research results was tested in a well-established dextran sodium sulfate (DSS)-induced colitis model in Nod-like receptor 3 (NLRP3)-deficient mice. This model offers a direct approach to determine whether MuAstV, as a component of the mouse microbiome, contributes to the issue of poor reproducibility in murine inflammatory bowel disease research. In this model, defective inflammasome activation causes loss of epithelial integrity, resulting in leakage of intestinal bacteria and colitis. Our goal was to determine whether MuAstV, which also may affect intestinal permeability, altered the onset or severity of colitis. Male and female mice (age, 8 to 12 wk) homozygous or heterozygous for an NLRP3 mutation were inoculated orally with MuAstV or mock-inoculated with media 3 or 20 d prior to being exposed to 2% DSS in their drinking water for 9 d. MuAstV infection alone did not cause clinical signs or histopathologic changes in NLRP3-/- or NLRP3+/- mice. No significant difference was seen in weight loss, clinical disease, intestinal inflammation, edema, hyperplasia, or mucosal ulceration between MuAstV- infected and mock-infected mice that received 2% DSS for 9 d. Therefore, MuAstV does not appear to be a confounding variable in the DSS colitis model in NLRP3 mice.

Murine Astrovirus Infection and Transmission in Neonatal CD1 Mice.

Murine astrovirus (MuAstV) is a recently identified, widespread infection among laboratory mice. Our goal was to determine the duration of MuAstV infection, susceptibility of pups, and efficacy of soiled-bedding sentinels and environmental monitoring. Eight CD1 dams and their litters of 3-d-old pups and 8 CD1 dams and their litters of 13-d-old mice were inoculated orally with MuAstV. Neither dams nor offspring demonstrated any clinical signs, and MuAstV had little to no effect on weight gain in pups. MuAstV RNA was detected in feces from 15 of the 16 dams through postnatal day (PND) 21, and 9 dams were still shedding MuAstV at PND 42. MuAstV RNA was highest in intestines of mice. Low levels of MuAstV RNA were sporadically detected in the spleen, liver, and kidney. MuAstV was detected in 97% of feces from 3- to 9-wk-old mice born to infected dams. Several weanlings became pregnant, and intestines from their pups were MuAstV-negative at PND 0 through 5. Weekly swabs of cages housing MuAstV-infected mice were MuAstV-positive through PND 42. Swabs of the rear exhaust manifold of the ventilated rack were MuAstV-positive at 21 through 56 d after inoculation. In addition, 98% of sentinels that received soiled bedding from dams and their litters and 83% of sentinels that received soiled bedding from weaned mice were MuAstV-positive. Feces from most sentinels exposed to soiled bedding that had been stored for 1, 2 or 3 wk before addition of the sentinels were MuAstV-positive.

Effect of Cage-Wash Temperature on the Removal of Infectious Agents from Caging and the Detection of Infectious Agents on the Filters of Animal Bedding-Disposal Cabinets by PCR Analysis.

Efficient, effective cage decontamination and the detection of infection are important to sustainable biosecurity within animal facilities. This study compared the efficacy of cage washing at 110 and 180 °F on preventing pathogen transmission. Soiled cages from mice infected with mouse parvovirus (MPV) and mouse hepatitis virus (MHV) were washed at 110 or 180 °F or were not washed. Sentinels from washed cages did not seroconvert to either virus, whereas sentinels in unwashed cages seroconverted to both agents. Soiled cages from mice harboring MPV, Helicobacter spp., Mycoplasma pulmonis, Syphacia obvelata, and Myocoptes musculinus were washed at 110 or 180 °F or were not washed. Sentinels from washed cages remained pathogen-free, whereas most sentinels in unwashed cages became infected with MPV and S. obvelata. Therefore washing at 110 or 180 °F is sufficient to decontaminate caging and prevent pathogen transmission. We then assessed whether PCR analysis of debris from the bedding disposal cabinet detected pathogens at the facility level. Samples were collected from the prefilter before and after the disposal of bedding from cages housing mice infected with both MPV and MHV. All samples collected before bedding disposal were negative for parvovirus and MHV, and all samples collected afterward were positive for these agents. Furthermore, all samples obtained from the prefilter before the disposal of bedding from multiply infected mice were pathogen-negative, and all those collected afterward were positive for parvovirus, M. pulmonis, S. obvelata, and Myocoptes musculinus. Therefore the debris on the prefilter of bedding-disposal cabinets is useful for pathogen screening.